Abstract
Keyhole limpet hemocyanin (KLH) of the mollusc Megathura crenulata is known to serologically cross-react with Schistosoma mansoni glycoconjugates in a carbohydrate-dependent manner. To elucidate the structural basis for this cross-reactivity, KLH glycans were released from tryptic glycopeptides and fluorescently labeled. Cross-reacting glycans were identified using a polyclonal antiserum reacting with soluble S. mansoni egg antigens, isolated by a three-dimensional fractionation scheme and analyzed by different mass spectrometric techniques as well as linkage analysis and exoglycosidase treatment. The results revealed that cross-reacting species comprise approximately 4.5% of released glycans. They all represent novel types of N-glycans with a Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc motif, which is known to occur also in schistosomal glycoconjugates. The tetrasaccharide unit is attached to the 3-linked antenna of a trimannosyl core, which can be further decorated by galactosyl residues, a xylose residue in 2-position of the central mannose and/or a fucose at the innermost N-acetylglucosamine. This study provides for the first time detailed structural data on the KLH carbohydrate entities responsible for cross-reactivity with glycoconjugates from S. mansoni.
Highlights
For potential application in anticancer therapy (9 –14)
Using enzymelinked immunosorbent assay (ELISA) as assay system, Keyhole limpet hemocyanin (KLH) glycopeptides were recognized by murine S. mansoni infection sera, the monoclonal antibody M2D3H reacting with the Fuc(␣1–3)GalNAc-epitope [23] and polyclonal antibodies raised against soluble egg antigen (SEA) of S. mansoni (Fig. 1)
In contrast to rabbit hyperimmune serum raised against KLH, antibody binding was mostly eliminated by mild HF treatment, which results in a release of fucose residues leaving the remaining carbohydrate chains largely intact [23]
Summary
KLH has been further demonstrated to exhibit a carbohydrate-based cross-reactivity with glycoconjugates from Schistosoma mansoni [15] allowing the diagnosis of S. mansoni [6, 7, 16], Schistosoma hematobium [17], and Schistosoma japonicum [18] infections by enzymelinked immunosorbent assay (ELISA). Regarding KLH cross-reactivity with schistosomal glycoconjugates, a fucose-containing carbohydrate epitope could be identified in S. mansoni glycolipids [22], which comprises terminal Fuc(␣1–3)GalNAc units [23] and is obviously shared by keyhole limpet hemocyanin. To provide a rationale for the recognition of this glycoprotein by using S. mansoni infection sera as well as monoclonal or polyclonal antibodies reacting with schistosomal glycoconjugates, we have performed a detailed structural analysis focusing exclusively on the serologically cross-reacting carbohydrate moieties
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